The present invention relates to a new electrospray ionization mass analysis apparatus and method thereof, wherein a sample solution is led to an electrospray ion (ESI) source and is ionized therein, and the ion generated therein is fed to an ion storage type mass spectrometer arranged in a highly vacuum space, where the ion is subjected to mass analysis.
In recent years there has been a remarkable growth in biological researches over diversified fields. Especially, protein, peptide and DNA play an extremely important role in the living body, and have been the objects of study by a great number of research workers.
Generally, these organic compounds derived from living organism occur in a very small amount in a complicated matrix. There has been a growing demand for extract a very small amount of these biological organic compounds from the living body and analyzing them using a mass spectrometer directly coupled with liquid chromatograph LC/MS apparatus) with a high degree of sensitivity. The LC/MS apparatus is an apparatus for separate a mixture with a liquid chromatograph (LC) and providing qualitative and quantitative analysis using axe2x80x94mass spectrometer (MS) with a high degree of sensitivity. Electrospray ionization (ESI) is typical ionization means used in the LC/MS. The ESI is an ionization technique used under atmospheric pressure and is known as providing soft and highly sensitive ionization. For this reason, this method has come to be used very often for biological analysis.
To ensure stable and highly sensitive measurement of a very small amount of components using the aforementioned ESI, some parameters must be optimized. One of these parameters is the flow rate for supply of the solution to the ESI ion source. To achieve highly sensitive measurement, the flow rate of the solution flowing through the ESI capillary tube must be kept within a certain range. In ESI, the optimum flow rate is said to lie in the range from 10 nL/min (10xe2x88x928 L/min) to several 1 xcexcL/min (10xe2x88x926 L/min). If a solution is fed into the ESI capillary tube at a flow rate higher or lower than this level, the ESI will become unstable and anticipated highly sensitive measurement will not be achieved.
The flow rate of a conventional LC often used for analysis of a mixture is in the range from several mL/min (several 10xe2x88x923 L/min) to several 100 xcexcL/min (several 10xe2x88x924 L/min). The flow rate of the semimicro LC is in the range from 100 xcexcL/min (several 10xe2x88x924 L/min) to several 10 xcexcL/min (several 10xe2x88x925 L/min). Since there is a big difference between the flow rate of the conventional LC and semimicro LC and the optimum ESI flow rate, both type of chromatography have been unable to be directly coupled with the ESI without solution being split. Needless to say, the splitting of solution will cause deterioration of sensitivity in measurement.
U.S. Pat. No. 4,861,988 discloses the art for improving the ESI to ensure that the aforementioned ESI can be applied to the conventional LC and semimicro LC of high flow rate. According to this art, the spray probe is improved to allow a stable spraying of a large volume of solution. As shown in FIG. 12, the ESI probe consists of two capillary tubes 30 and 32 having different outer diameters. The first capillary tube 30 having a smaller diameter is inserted into the second capillary tube 32 having a larger inner diameter. This configuration provides a coaxial ESI probe. A high voltage of 3 to 4 kV is supplied from the DC high voltage power supply 5 and is applied to the first capillary tube 30. Sample solution is led into the first capillary tube. Nitrogen gas is fed to the space between the first and second. Sample solution is released into the atmosphere as minute charged droplet 6 by the mechanical force and action of electric field. The charged droplet is further pulverized by mechanical crushing due to the flow of gas and evaporation of solution. Ion is released into the atmosphere in the final stage. The generated ion is introduced into a high-vacuum mass spectrometer.
This method has brought about a drastic increase in the flow rate to be introduced into the ESI probe. The increase is from 1 mL/min (10xe2x88x923 L/min) to 100 xcexcL/min (10xe2x88x924 L/min). This is just applicable to the flow rate of the conventional LC (several mL/min to several 100 xcexcL/min) and semimicro LC (several 100 xcexcL/min to several 10 xcexcL/min). This method has come to be called xe2x80x9cIon Sprayxe2x80x9d or xe2x80x9cPneumatic Assisted Electrosprayxe2x80x9d.
U.S. Pat. No. 5,504,329 discloses another art of improving the ESI permitting measurement of still further minute components with high sensitivity. The art disclosed therein was later called Nanospray technique. After the tip of an extra-fine capillary tube made of glass having an outer diameter of about 0.2 mm and inner diameter of about 0.03 mm has been elongated by a burner or sharpened by etching, the nozzle tip is gold plated. The D.C. voltage of about 1 kV supplied from the high voltage source is applied to the tip of the nozzle. The flow rate of a sample solution from a nanospray device ranges from is several nL/min (several 10xe2x88x929 L/min) to 10 nL/min (several 10xe2x88x928 L/min). Measurement for more than one hour was enabled by only the sample sucked into the nanospray spray capillary tube. Accordingly, this nanospray technique has come to be used in combination with extra-low flow rate chromatography in CE (Capillary Electrophoresis); further, it has come to be used for extremely highly sensitive measurement of isolated components. The nanospray technique has enabled ESI measurement in the range of flow rate below 10 nL/min.
Table 1 shows the ESI art, improved ionization arts, optimum flow rate and compatible chromatography:
The advent of Ion Spray, ESI and Nanospray has enabled selection of an ion source suited to each one of various types of chromatography. However, as shown in Table 1, there is no ESI ion source that is best suited to the micro LC from 10 xcexcL/min to 1 xcexcL/min. For this reason, the researchers have to use the ESI or Ion Spray for the micro LC, based on the understanding that the sensitivity and stability are not satisfactory.
For the micro LC, the flow rate of mobile phase is from 10 xcexcL/min to 1 xcexcL/min and the required amount of sample solution can also be reduced to the level as small as nL(10xe2x88x929 L/min). So its range of application is rapidly expanding to cover the field of analyzing the biological component. Needless to say, there has been a growing demand for ionization technique optimum to this field.
The object of the present invention is to provide an electrospray ionization mass analysis apparatus and the method thereof provided with an ESI ion source that can be directly coupled to the micro LC.
The present invention provides an electrospray ionization mass analysis apparatus wherein:
a sample solution is led into a capillary tube under atmospheric pressure,
voltage or electric field is applied to the tip of this capillary tube, thereby allowing an ion to be generated by an electrospray ion source provided therein, and
generated ion is led to and stored in an ion storage type spectrometer disposed in a vacuum chamber so that a mass spectrum is obtained by subsequent mass sweeping. This electrospray ionization mass analysis apparatus is characterized in that
the voltage formed by AC voltage superimposed on the DC voltage or the electric field formed by AC electric field superimposed on the static electric field is applied to the tip of the aforementioned capillary tube, whereby electrospray ionization is carried out.
The present invention provides an electrospray ionization mass analysis apparatus wherein:
a sample solution is led into a capillary tube; high voltage is applied to the tip of this capillary tube, thereby allowing a spray ion flow of the aforementioned solution to be generated by an electrospray ion source provided therein; and
the ion flow generated by this ion source is led to an ion storage type spectrometer disposed in an vacuum chamber where it is subjected to mass sweeping, and the swept ion is detected by a detector so that a mass spectrum is obtained. This electrospray ionization mass analysis apparatus is characterized by comprising a high voltage DC power source for application of the aforementioned high voltage formed by AC voltage superimposed on the DC voltage, and a AC power source.
The present invention provides an electrospray ionization mass analysis apparatus wherein:
a sample solution is separated by a micro liquid chromatograph;
the sample solution separated by the micro liquid chromatograph is led into a capillary tube;
high voltage is applied by a high voltage power source connected between the tip of the capillary tube and a counter electrode having an aperture, thereby allowing a spray ion flow to be generated to flow from the tip of the capillary tube toward the aperture by an electrospray ion source provided therein; and
the ion flow generated by this ion source is sequentially led from the aperture to a skimmer cone and ion guide disposed in an vacuum chamber, and then to the ion storage type spectrometer where it is subjected to mass sweeping and the swept ion is detected by a detector so that a mass spectrum is obtained. This electrospray ionization mass analysis apparatus is further characterized in that the aforementioned high voltage power source comprises a high voltage DC power source for application of DC voltage and an AC power supply for application of AC voltage, wherein the voltage is formed by the aforementioned AC voltage superimposed on the aforementioned DC voltage.
The present invention provides an electrospray ionization mass analysis apparatus characterized by one of the following configurations; (1) the aforementioned skimmer cone, ion guide and ion storage type mass spectrometer are each disposed integrally in each vacuum chamber, which is provided with a vacuum pump; (2) an XYZ3 axis positioner for setting the spray ion flow with respect to the aforementioned capillary tube is connected; and (3) the ion storage type mass spectrometer is an ion trap mass spectrometer or an ion cyclotron resonance (ICR) mass spectrometer.
The present invention provides an electrospray ionization mass analysis apparatus wherein:
a sample solution is led into a capillary tube under atmospheric pressure;
high voltage is applied to the tip of this capillary tube, thereby allowing a spray ion flow of the aforementioned solution to be generated; and
the generated ion flow is led to an ion storage type spectrometer disposed in an vacuum chamber where it is subjected to mass sweeping, and the swept ion is detected by a detector so that a mass spectrum is obtained. This electrospray ionization mass analysis apparatus is characterized in that the voltage formed by AC voltage superimposed on the DC voltage or the electric field formed by AC electric field superimposed on the static electric field is applied to the tip of the aforementioned capillary tube.
The present invention provides an electrospray ionization mass analysis apparatus wherein:
a sample solution is separated by a micro liquid chromatograph;
the sample solution separated by the micro liquid chromatograph is led into a capillary tube;
high voltage is applied to the tip of the capillary tube, thereby allowing a spray ion flow to be generated to flow from the tip of the capillary tube; and
the generated ion flow is sequentially led to a skimmer cone and ion guide disposed in an vacuum chamber, then to the ion storage type spectrometer where it is subjected to mass sweeping, and the swept ion is detected by a detector so that a mass spectrum is obtained. This electrospray ionization mass analysis apparatus is characterized in that the voltage formed by AC voltage superimposed on the DC voltage or the electric field formed by AC electric field superimposed on the static electric field is applied to the tip of the aforementioned capillary tube.
The present invention provides an electrospray ionization mass analysis apparatus characterized by one of the following configurations; the aforementioned DC voltage and AC voltage or the intensity of static electric field and AC electric field can be set and controlled from the outside; when the aforementioned spray ion flow is in the positive ion measurement mode, the polarity of the aforementioned DC voltage or static field is positive; and when the aforementioned spray ion flow is in the negative ion measurement mode, the polarity of the aforementioned DC voltage or static field is negative; the aforementioned AC voltage is 100% or less of the DC voltage, or the AC electric field is 100% or less of the static electric field, preferably, in the range from 10 to 65%, more preferably in the range from 15 to 50%; the frequency of the aforementioned AC voltage or AC electric field is 1 kHz or less, preferably in the range from 31 to 330 Hz, more preferably in the range from 50 to 300 Hz; the flow rate of the solution led into the capillary tube under atmospheric pressure is 50 xcexcL/min or less, preferably 20 xcexcL/min or less; and high voltage applied to the tip of the capillary tube is such that the sample solution is formed in a Taylor cone shape at the outlet of the capillary tube.
The present invention according to any one of the aforementioned methods is characterized in that the aforementioned ion storage type spectrometer is an ion trap type spectrometer, and the following steps are incorporated:
(1) Preparatory step wherein the voltage formed by DC voltage superimposed on AC voltage is applied to the capillary tube. Voltage of the same polarity as that of ion is applied to the ion gate electrode arranged on the front stage of the ion trap type spectrometer so that ion will not be led into the ion trap. The main high frequency voltage applied to the ring electrode constituting the ion trap electrode is reset to zero to remove all the ions in the ion trap.
(2) Ion introduction and storage step wherein voltage of the polarity reverse to that of ion is applied to the ion gate electrode so that ion can be led into the ion trap. The main high frequency voltage is applied to the ring electrode so that the ion in a predetermined mass range is led into the ion trap, and ion storage is carried out for a predetermined period of time level out the ion current.
(3) MS/MS step wherein voltage of the same polarity as that of ion is applied to the ion gate electrode to block introduction of the ion. The ion in the aforementioned ion trap is selected and collision dissociation (CID) is carried out. This step can be omitted according to the purpose of each analysis.
(4) Mass spectrum acquisition step wherein the main high frequency voltage applied to the ring voltage is swept and the ions in the ion trap are discharged out of the ion trap sequentially in the order of the mass. Ion current value is detected by a detector and the detected signal is fed to a control data processor, where the mass spectrum is obtained.
Namely, the mass analysis using the ESI according to the present invention is carried out as follows: Voltage of several kilovolts is applied between a metallic capillary having an inner diameter of about 0.1 mm and a counter electrode arranged at some distance (about several tens of mm) away therefrom. When a sample solution is led to the metallic capillary and a high voltage is applied, the liquid in the capillary is dielectrically polarized at the capillary outlet by a high electric field formed on the tip of a metallic capillary. In the positive ionization mode, positive electric charge is induced on the liquid surface, while in the negative ionization mode, negative electric charge is induced on the liquid surface.
As a result, a conical liquid called Taylor cone is pulled out into the atmosphere from the capillary outlet by electric field. If electric field is stronger than the surface tension at the tip of the Taylor cone, electrically charged extremely fine droplets are released into the atmosphere from the tip of the Taylor cone. In conformity to electric field, the generated charged droplets fly in the atmosphere toward a counter electrode to repeat collision with molecules in the atmosphere.
This allows charged droplets to be mechanically crushed, and evaporation of solvent from the droplet surface is promoted so that charged droplets are quickly pulverized. In the final stage, ions in charged droplets are released into the atmosphere. The ion flies in the atmosphere toward a counter electrode and is led into a highly vacuum mass spectrometer through a capillary tube or aperture arranged in the counter electrode where it is subjected to mass analysis.
In the present invention using the micro LC, the flow rate of mobile phase is several tens of 10 xcexcL/min to 1 xcexcL/min and the required amount of sample solution to be supplied is as small as nL(10xe2x88x929 L/min). Accordingly, its application can be expanded to the field of analysis of biological related components where trace quantities of components are handled.